The aggressiveness of macrophages in fighting infection or killing cancer cells is regulated by macrophage activation. Activation in vitro is called "priming". Primed macrophages release more microbicidal oxygen radicals. Macrophages are primed by bacterial factors like lipopolysaccharide (LPS) and muramyl peptides, by cytokines like interferon-gamma, and by lipid mediators like platelet-activating factor. Earlier, we showed in mice that the activator muramyl dipeptide enhanced non-specific resistance to lethal infections. We recently found that priming of monocytes was inhibited by the serine protease inhibitor AEBSF. This suggests that, in addition to kinases and other signal transduction elements, proteases are essential in macrophage activation. Objective: Elucidate the role of proteases in macrophage activation. Specific Aim 1: Identify the negative regulator of priming. Hypothesis: Priming is negatively regulated by a protein, whose stability, or continuous synthesis, is required to keep monocytes unprimed. This regulator protein can be identified in two-dimensional gels, comparing monocytes q LPS q AEBSF. Specific Aim 2: Identify the protease responsible for inactivating the negative regulator of priming. Hypothesis: Priming is prevented by a monocyte protein that acts as a negative regulator; removal of the regulator by an endogenous protease causes priming. The protease is inactivated by AEBSF, which blocks priming. The protease can be identified by tagging it with radiolabeled AEBSF. Specific Aim 3: Investigate the mechanism by which LPS inactivates or blocks the synthesis of the negative regulator of priming. Hypothesis: LPS and other primers activate the protease, or induce synthesis of the protease, that cleaves the negative regulator of priming. Covalent modification or synthesis of the protease, or removal of an inhibitor, can be observed in response to LPS. Specific Aim 4: Determine how the protease and its substrate interact with other signal elements like kinases and phospholipases. Hypothesis: The substrate of the protease (the negative regulator) is an inhibitor of a phospholipase or kinase that is essential for macrophage activation. Significance: Knowledge of mechanisms of macrophage activation will help us to fight infection, which will increase due to aging of the population, more immuno-compromised patients, and antibiotic-resistant microbes.